Abstract Increasing evidence indicates that clinical responses to immune checkpoint blockade (ICB) in solid tumors are mediated by T-cell recognition of ?neoantigens? created when peptides containing amino acids altered by cancer specific mutations are presented on major histocompatibility complex (MHC) proteins. Thus, strategies to augment such T-cell responses independent of, or concurrent with ICB, could be of therapeutic benefit. Prior work on neoantigen-reactive T cells has focused on CD8+ T cells which directly recognize antigens presented on class I MHC expressed by tumor cells. However, there is emerging evidence that CD4+ T cell responses directed against neoantigens presented by class II MHC on professional antigen presenting cells (APC) are common, contribute to tumor rejection in mouse models, and are effective in cancer patients treated with adoptively transferred tumor infiltrating lymphocytes. I recently discovered CD4+ T-cells specific for recurrent driver mutations in BRAF, KRAS, Her2 and EGFR in cancer patients, and have cloned the specific T cell receptors (TCRs) to use for adoptive immunotherapy of patients that express these mutations and have the correct MHC alleles. Driver mutations make ideal immunotherapy targets because, unlike most neoantigens that are random and patient specific, they are positively selected for clonal and homogenous expression and are found in multiple patients. We and others have shown that driver mutation-specific CD4+ T cell responses are commonly present, and that these T cells can be expanded locally in tumors, suggesting they recognize tumor antigens in vivo. In mouse models, tumor antigen-specific CD4+ T cells can mediate tumor rejection through direct destruction of tumor cells, activation of innate immune cells, and stimulation of CD8+ T cell responses, but the mechanisms of action by CD4+ T cells in human antitumor immunity are largely unknown. High dimensional single cell analysis of human tumors has shown heterogeneity of CD4+ T cells with regards to activation, exhaustion, cytolytic potential, and differentiation states associated with stimulation versus suppression of cellular immune responses, but which of these populations contain the small subset of neoantigen specific CD4+ T cells is unknown. In specific aim 1 we will address the frequency, activation, and differentiation state of neoantigen specific CD4+ T cells in tumors to better understand whether and how these cells might contribute to antitumor immunity. We will then use our discovery of a TCR that can redirect the specificity of CD4+ T cell to the common BRAF V600E mutation to ask whether adoptive cell transfer of neoantigen-specific CD4+ T cells can be safe in a first in human phase I clinical trial in specific aim 2. This trial will give the opportunity for asking whether adoptively transferred CD4+ T cells can mediate therapeutic effects and address potential mechanisms for CD4+ T cells to mediate antitumor immunity. If successful, this approach could be applied to additional targets in larger number of patients.